The present invention relates to methods of reducing chemical fertilizer usage and greenhouse gas nitrous oxide (N2O) emission and to methods of improving plant growth rate and seed productivity in agriculture through the application of a novel artificially manufactured formula containing a nitrogen-fixing bacterium that efficiently colonizes non-legume plants in aerial parts and the root system. The bacteria inocula and methods are particularly suitable for plants in the genera Jatropha, Sorghum, Gossypium, Elaeis, Oryza, Ricinus and Manihot. 
The rapid rise in fossil fuel price, the fast diminishment of global fuel and the concern about rapid global warming resulted from the accumulation of atmospheric greenhouse gas have served as the three catalysts in the recent biofuel boom (Chang, 2007). The environmental benefit of biofuel consumption is attributed from the widely believed outcome of reduced greenhouse gas (CO2) emission.
Recently, a new issue that has been brought up for debate regarding the benefit of biofuels. Nitrous oxide is produced naturally in the soil during the microbial processes of nitrification and denitrification. A significant percentage of the nitrogen fertilizer used in biofuel production is converted to reactive nitrogen N2O, a greenhouse gas which has 310 times the ability to trap heat in the atmosphere (Barton and Atwater, 2002). The benefit of CO2 mitigation through biofuel consumption will be cancelled out if the use of N-fertilizer is not controlled (Galloway et al., 2008; Melillo et al, 2009; Crutzen et al., 2008).
Application of nitrogen fertilizer has become an essential practice in modern agriculture as it is vital to maintain competitive crop productivity. It has been well known that legumes require much less input of nitrogen fertilizer owing to the presence of nitrogen-fixing structure called nodules, in which nitrogen-fixing microorganisms (diazotrophs), mostly belonging to species in the Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium genera (Jourand et al., 2004; Kaneko et al., 2000; Stacey et al., 1991; Gottfert et al., 2001), form a mutually beneficial symbiotic relationship with the bacteria supplying nitrogen source to the plants while drawing carbon source from the plants cells (Long, 1996; Young and Johnston, 1989). Some exception has been found recently. For example, Methylobacterium nodulans is the root nodule-inducing agent for some species in the genus Crotalaria, a leguminous plant (Renier et al., 2011; Jourand et al., 2005) and nitrogen-fixing nodules may develop in stem tissues of some legumes (Eaglesham and Szalay, 1983; Dreyfus and Dommergues, 1981). Frankia, Gram-positive soil bacteria also induce the formation of nitrogen-fixing root nodules in a few species in the Rosaceae family (Moir et al., 2011).
The formation of symbiotic nodules involves with complex genetic and chemical interactions between the diazotroph and host. For example, the symbiosis between Sinorhizobium meliloti and its plant hosts begins when the plant secretes an array of betaines and flavonoids into the rhizosphere: 4,4′-dihydroxy-2′-methoxychalcone, chrysoeriol, cynaroside, 4′,7-dihydroxyflavone, 6″-O-malonylononin, liquiritigenin, luteolin, 3′,5-dimethoxyluteolin, 5-methoxyluteolin, medicarpin, stachydrine, trigonelline. These compounds attract S. meliloti to the surface of the root hairs of the plant where the bacteria begin secreting nodulation factor (Peters et al., 1986; Maxwell et al., 1989). As a consequence, symbiotic nodule-forming nitrogen-fixation is found almost exclusively in legume species. Bradyrhizobia and rhizobia share characteristics with plant growth promoting rhizobacteria (PGPR). Nodule inducing bacteria, like other PGPR, are capable of colonizing the roots of non-legume plants (Antoun et al., 1998).
Diazotrophs have been found in free-living bacteria, among which Azotobacter vinelandii is the best studied. An increasingly number of diazotrophic species, e.g., Azospirillum, Herbaspirillum, Burkholderia, Gluconacetobacter, have been reported to form atypical symbiotic relationship with plants (Zehr, 2011; Radiers et al., 2004; Pedraza, 2008). They often grow on the surface root system (rhizobacteria) although some are able to infect plant tissues (endophytic bacteria) and perform nitrogen fixation, which is also able promote plant growth. For example, U.S. Pat. No. 7,393,678 B2 describes strains of Klebsiella pneumonia, which colonize the root surface (Liu et al., 2011), are able to promote growth of cereals (wheat and corn) either in the presence or absence of chemical nitrogen application. However, only specially isolated mutant strains of Klebsiella pneumonia were able to perform satisfactory plant growth promoting function as the nitrogen-fixation activity is low in majority of strains. Perhaps, the best examples of endophytic nitrogen-fixation can be found in sugarcane and wild rice, with Herbaspirillum, Gluconacetobacter, Enterobacter, Azospirillum, Swaminathania and Acetobacter being the possible contributors of nitrogen-fixing species (Pedraza, 2008; Boddey et al., 1995; Baldani et al., 2002; Elbeltagy et al., 2001; Saravanan et al., 2008). Photosynthetic Bradyrhizobia are the natural endophytes of the African wild rice Oryza breviligulata while the intercellular colonization and growth-promoting effects of a Methylobacterium sp. was observed in common rice Oryza sativa L. Cv CO-43 although the later was believed to resulted from phytohormone secretion of the bacterium (Senthilkumar et al., 2009; Chaintreuil et al., 2000).
A study of Azospirilla indicates that, similar to nodulation and nitrogen-fixation in legumes, the endophytic colonization process is genetically controlled and exopolysaccharide production inhibits both endophytic colonization and nitrogen fixation in common wheat seedlings (Kennedy et al., 1997). In addition, a mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli (Borthakur et al., 1986). Therefore, the ability of a diazotroph to promote plant growth via nitrogen-fixation is not predictable from its performance in other plants.
Jatropha curcas is a small woody plant belonging to the Euphorbiaceae family. Several unique characteristics make it an ideal plant for biodiesel production (Fairless, 2007; Gaydou et al., 1982; Openshaw, 2000). These include the ability to grow on marginal land; low requirement for water; a non-food crop status and fast oil production in 0.5-2 years after planting compared to more than 3 years for oil palm. Accordingly, several Asian countries, particularly Indonesian and India, have made ambitious plans to promote jatropha plantation. Several other plants have also attracted strong interest as alternative crop for biofuel production. These include castor bean, sorghum, and sweet sorghum.
As Jatropha is targeted to marginal land where soil nutrient is low, the requirement for nitrogen fertilizer will be higher than other crops. Therefore, any technology that reduces nitrogen fertilizer usage will be highly desirable. Today there is little research on naturally occurring diazotrophs in Jatropha nor the application of these microbes for improvement of plant productivity. Similar situations can be found in other crops, such as Sorghum, castor bean and cassava.